A gene disruption method has been used as a tool to analyze genome of rice (Oryza sativa). To disrupt genomes of rice, a method to activate a segregation factor by mating an individual, wherein a transposase gene (transposition enzyme) is tranduced as an activator, with an individual, wherein a segregation factor is introduced; a method to use T-DNA; and a method to use retrotransposon have been known. However, analysis of spontaneous mutants of rice has not served to find active transposons (Supplementary volume of Cell Engineering, Plant cell engineering series 14, “Plant genome research protocols” 2000, February, Shujun press Co.).
On the other hand, a genome sequencing project is determining the nucleotide sequences of many plants as well as rice and the results are databanked. Furthermore, transposon genes, mobile genes in animal and plant, have, to some extent, unique nucleotide sequences, whose information has enabled research on wider applications of transposons. However, enough elucidation has not been demonstrated. Additionally, a nucleotide sequence, assigned to a putative transposon gene has been found in a mutated rice induced by γ-irradiation (Tetsuya Nakazaki et al., “Polymorphic insertion of transposon-like sequence in mutable slender-granule gene slg locus” Japanese Society of Breeding 100th Conference, Autumn meeting, 2001, October).
Problems to be Solved by the Invention:
The inventors discovered an inverted repeated sequence characteristic to transposon genes in rice genome nucleotide sequence under investigation. Examining various tests on the possible transposon nucleotide sequence, the inventors confirmed that the nucleotide sequence is ascribed to a transposon gene (nonautonomous transposon).
Furthermore, the inventors discovered autonomous transposon genes on the basis of the nonautonomous transposon gene. Moreover, the inventors identified transposase genes, which enable to transpose the transposon gene.
Means of Solving the Problems:
The inventors, starting from chromosome No. 1, examined long terminal repeats (LTR) in rice genome sequence, which is registered seriatim in database. The inventors noticed a LTR on the clone, accession number AP002843, as shown in Table 1, investigated the sequence in detail and discovered the inverted repeat sequence characteristic to a transposon gene at the site of 144459th–144473rd and 144874th–144888th bases at adjacent downstream of LTR (FIG. 1, SEQ ID NO: 6). As disclosing in the example shown later, the inventors confirmed that the nucleotide sequence (SEQ ID NO: 1) located between the inverted repeats is a nonautonomous transposon gene, markedly transposable by such artificial treatment as anther culture.
TABLE 1AP002843 Oryza sativa genomic DNA, chromosome 1, PA ACCESSIONAP002843NCBI SRS Genome-Net ORGANISMOryza sativaNCBI SRSLOCUSAP002843148762 bp  DNA  PLN   26-JAN-2001FEATURESLocation/Qualifierssource  1..148762/chromosome=“1”/clone=“P0407B12”/cultivar=“Nipponbare”/organism=“Oryza sativa”/sequenced_mol=“DNA”LTR  139482..139690/note=“5′ LTR”CDS  139739..144052/gene=“P0407B12.28”/note=“probably inactive due to frameshifts in CDS”/note=“pseudogene, similar to Oryza longistaminataprobable gag/pol polyprotein U72725”/pseudoLTR  144047..144255/note=“3′ LTR”CDS  join(144653..144692, 145148..145311)/codon_start=1/gene=“P0407B12.29”/note=“hypothetical protein”/protein_id=“BAB17191.1”/translation=“MRRSHGGGGRKRSVPSSSHPEKKAIDRIKREDAGRRAGRVSLVQPLAAFPATDGGGGGGLARLLRWW”   (SEQ. ID NO: 28)
Then, the inventors searched for homologous sequences using the nonautonomous transposon gene (SEQ ID NO: 1) by Blast search. Most of the results of the search lead to the nonautonomous transposon gene (SEQ ID NO: 1) itself, but additionally, accession numbers AP004236 and AP003968, which were expected as transposon genes, were found. Comparing the nucleotide sequence of AP004236 and AP003968, the inventors found that these are cloned on chromosome No. 6 and are the sequence of the identical overlapped region.
Therefore, the homology between 1st–253rd bases of the nonautonomous transposon gene (SEQ ID NO: 1) and 89360th–89612nd bases of AP004236 was 252/253 (99%) and that between 254th–430th bases of the nonautonomous transposon gene (SEQ ID NO: 1) and 94524th–94700th bases of AP004236 was 177/177 (100%). They are well-conserved sequences.
Both nonautonomous transposon gene (SEQ ID NO: 1, 430 bp) and the transposon (SEQ ID NO: 2, 5341 bp) have inverted repeats of 15 bp and TTA and TAA are recognized and inserted. Open Reading Frame (ORF) was searched on the basis of SEQ ID NO: 2 (5341 bp) and two kinds of putative ORFI and ORFII were obtained.
Open Reading Frame (ORF) was searched on the basis of Sequence Number 2 (5341 bp) and two kinds of putative ORFI and ORFII were obtained.
The structure of the transposon gene comprising nucleotide sequence of SEQ ID NO: 2 is shown in the upper diagram of FIG. 9. The nonautonomous transposon gene (SEQ ID NO: 1, 430 bp) is located at 1st–253rd and at 5165th–5341st bases, ORFI is located at 1526th–1914th bases and at 1939th–2663rd bases and ORFII is located at 3190th–4557th bases.
Furthermore, to obtain similar genes to the gene comprising nucleotide sequence of SEQ ID NO: 2, the inventors performed homological searches using nucleotide sequence of SEQ ID NO: 2 as Query (DNA for homological search) of Blast search and found accession numbers AP004753 (chromosome No. 2) and AP003714 (chromosome No. 6) as well as the sequence of SEQ ID NO: 2 itself. These two clones have the identical nucleotide sequence (SEQ ID NO: 3) and located on different chromosomes (several copies). Since the nucleotide sequence is present also in indica (a cultivar of rice), the gene must be conserved in many cultivars, from japonica to indica. The nucleotide sequence of SEQ ID NO: 3 (5166 bp) has inverted repeats, as the sequence of SEQ ID NO: 2 has, and TAA (3 bp) was also recognized and inserted.
Open Reading Frame (ORF) was searched on the basis of the sequence of SEQ ID NO: 3 and ORFI and ORFII were obtained which may code two kinds of proteins. The structure of transposon gene comprising nucleotide sequence of SEQ ID NO: 3 is shown in the lower diagram of FIG. 9. The nonautonomous transposon gene (SEQ ID NO: 1, 430 bp) is located at 1st–170th and at 5092nd–5166th bases, however, the homological nucleotide sequence to the nonautonomous transposon gene (SEQ ID NO: 1, 430 bp) is disappeared in the middle. ORFI is located at 1630th–2652nd bases and ORFII is located at 2959th–4407th bases. The nucleotide sequence of SEQ ID NO: 3 was compared between japonica (AP004753 and AP003714) and indica (Scaffold6962) and the homology of more than 90% was confirmed as shown in FIG. 10. Additionally, the inventors examined the mutation frequency of nonautonomous transposon gene (SEQ ID NO: 1, 430 bp) in indica, and confirmed that the sequence homology was more than 95% as shown in FIG. 11.
The inventors have no idea on the function of ORFI in SEQ ID NOS 2 and 3, for the moment.
To examine whether ORFII encodes transposase (transposition enzyme) or not, the inventors checked whether the amino acid sequence of ORFII shares a conservative region with that of known transposase gene. The amino acid sequences of ORFII in SEQ ID NO: 2 and in SEQ ID NO: 3 are shown as SEQ ID NOS 4 and 5, respectively. The alignment of amino acid sequences of these two ORFII (SEQ ID NOS 4 and 5) is shown in FIG. 12 and the homology of these sequences was more than 75% (77%). These two amino acid sequences have DXG/AF/F motif and YREK motif (SEQ ID NO: 39) (Q. H. Le, K. Turcotte and T. Bureau, Genetics 158: 1081–1088 (2001)), then it is concluded that thee belong to IS transposase family.
Also, the homology of the nucleotide sequences of ORFII in SEQ ID NO: 2 and that in SEQ ID NO: 3 was more than 75% (79.3%).
Therefore, the present invention is a transposon gene of rice consisting of a nucleotide sequence which is at least 95% homological to SEQ ID NO: 1. The DNA being at least 95% homological to SEQ ID NO: 1 is considered to be functional as nonautonomous transposon, which is transposable by anther culture or by the treatment with chemical agents. Also, the present invention is the transposon gene of rice, wherein enhancers or promoters are inserted.
Furthermore, the present invention is the transposon gene of rice, whose nucleotide sequence is at least 90% homological to the nucleotide sequence of SEQ ID NO: 2 or 3. The DNA being at least 90% homological to the nucleotide sequence of SEQ ID NO: 2 or 3 is considered to be functional as an autonomous transposon gene, which is transposable by anther culture or by the treatment with chemical agents.
Also, the present invention is the transposase gene of rice, whose DNA being at least 75% homological to the nucleotide sequence of 3190th–4557th bases off SEQ ID NO: 2 or the nucleotide sequence of 2959th–4407th bases of SEQ ID NO: 3. The DNA being at least 75% homological to these nucleotide sequences is considered to be functional as the gene, which enables transpose the transposons.
Also, the present invention is the transposase gene encoding a protein consisting of an amino acid sequence of SEQ ID NO: 4 or 5 or an amino acid sequence wherein one or several amino acids are deleted, substituted or added in said amino acid sequence. Also, the transposase could be a transposon gene of rice, whose amino acids sequence is at least 75% homological to SEQ ID NO: 4 or 5.
Moreover, the present invention is the transposase gene encoding this protein. Also, the present invention is the plasmid containing any one of said transposon genes. Still furthermore, the present invention is the plasmid containing promoters and anyone of said transposase genes. Such binary vector as Ti plasmid and pBI-121 plasmid can be used for the purpose. 35S promoter of cauliflower mosaic virus, heat shock promoter, chemotaxis promoter and others can be used for the purpose of this invention. There are no restrictions on the method of incorporation of promoters and said genomes and general method of genetic engineering can be applied.
Also, the present invention is the transfomants, wherein any of said transposon genes are transduced. Preferably, plants, especially, rice, barley, wheat or maize are used as the host. To transform these plants, using general method of genetic engineering, we can insert these genomes into said plasmid and transform the plants.
Still moreover, the present invention is the transfomants, wherein promoters and said transposase genes are transduced. Other transposon genes can be transduced, if necessary. Said promoters can be used for the present purpose. Preferably, plants, especially, barely, wheat or maize are used as the host. To transform these plants, using general method of genetic engineering, we can insert said genomes into said plasmid and transform the plants.
Also, the present invention is the method for transposing any of said transposon genes, comprising subjecting said transformants to anther culture or treating any of the transformants with a chemical agent.
Furthermore, the present invention is the plant or the seed, which is transformed by the transposition of said transposon genes by any one of said methods. Preferably, rice or barley, a vicinal species of rice, wheat or maize is used as the plant.
Also, the present invention is a method for determining the integrated region of transposon gene, which comprises the steps of transposing any one of said transposon gene by any one of the above methods, extracting DNA from the plant obtained by the previous step, digesting said DNA by a restriction enzyme with no cutting sites inside the transposon gene, ligating said DNA fragments obtained by the previous step, conducting PCR for said DNA fragments obtained by the previous step, and determining the nucleotide sequence of said PCR products obtained by the previous steps. The primers of said PCR involve the oligonucleotides, which comprises 10 consecutive bases, preferably 10˜20 consecutive bases, more preferably 10˜15 consecutive bases in the nucleotide sequence from the 5′-end of SEQ ID NO: 1; and the oligonucleotide, which comprises 10 consecutive bases, preferably 10˜20 consecutive bases, more preferably 10˜15 consecutive bases in the nucleotide sequence from the 3′-end of SEQ ID NO: 1, or the oligonucleotides, comprising the nucleotide sequences complementary to said sequences. Since the origonucleotide comprising 10˜15 consecutive bases from the 5′-end of the nucleotide sequence overlaps with that comprising 10˜15 consecutive bases in the nucleotide sequence from the 3′-end of that of SEQ ID NO: 1, we can use single kind of primer, if the oligonucleotide bases comprises less than 15 consecutive bases. In other words, in this case, we can use the oligonucleotide comprising 10˜15 consecutive bases in the nucleotide sequence from the 5′-end of SEQ ID NO: 1 or the oligonucleotides complementary to said nucleotide sequences as the PCR primer. In this way, identification of the integration site of the transposon enables to find the disrupted genomes.